Radiation-induced neoplastic transformation of C3H10T1/2 cells is suppressed by ascorbic acid

X-ray induced transformation of C3H10T1/2 cells was suppressed in a concentration-dependent manner by administration of ascorbic acid after irradiation (0.1-20 micrograms/ml for the first week) in the culture medium. The dose-response curve was shifted about 60% downward and was slightly steeper in the presence of ascorbic acid (5 micrograms/ml for the first week) than in its absence. The 1-week treatment procedure revealed that cells initiated by radiation remained susceptible to ascorbic acid until the time of morphological phenotype expression. The neoplastically transformed phenotype expressed after incubation for 8 weeks could no longer be suppressed by ascorbic acid even after culture transfer. Similarly, the neoplastically transformed phenotype suppressed for 8 weeks by ascorbic acid treatment was not subsequently expressed in the absence of ascorbic acid. On the basis of the oxygen-detoxifying nature of ascorbic acid, we postulated that expression of the neoplastically transformed phenotype is promoted by reactive oxygen species and peroxy radicals generated in cells during the whole assay period. The data may be useful as a guide for chemopreventive efforts against radiation carcinogenesis.     

Colony transformation in C3H 10T1/2 cells: stepwise development of neoplastic change in vitro

This report demonstrates that low passage C3H 10T1/2 cells treated with the carcinogens benzo(a)pyrene or diepoxybutane are transformed morphologically as colonies in as little as 14 d after carcinogen treatment. A transforming dose-response curve is achieved but the frequency of transformation is less than half the expected for 38 d foci, compared on the basis of percent transformants per cell plated. Anchorage-independent cell growth, plating efficiency, doubling time, cell density, and modal chromosomal number were examined from transformed colonies and foci. The data from colony transformants show progressive alteration of these in vitro expressions of neoplastic character with continued subcultivation, consistent with the multistep hypothesis of carcinogenesis. Early in vivo data obtained from one colony-derived transformed cell line show tumorigenesis in irradiated mice within 13 wk of implantation. With continued in vivo passage, tumors were observed in 4 to 6 wk.     

Induction of neoplastic transformation in C3H/10T1/2 cells by 2.45-GHz microwaves and phorbol ester

C3H/10T1/2 cells were exposed to 2.45-GHz microwaves for 24 h and/or 1.5 Gy of 238-kVp X rays at 3.75 Gy/min. Transformation frequency and cell survival were measured with or without postirradiation addition of the tumor promoter tetradecanoyl-phorbol-13-acetate (TPA) at 0.1 microgram/ml. We previously reported (Carcinogenesis 6,859-864, 1985) an enhancement of transformation frequency when 10T1/2 cells exposed to a special sequence of microwaves and X rays were subsequently cultured in TPA. The same sequence of microwaves and X rays without promotion resulted in a transformation response similar to that induced by X rays alone. We now report statistically significant (at P greater than 0.999) enhancement of transformation response by TPA in cells exposed to 2.45-GHz microwaves (SAR = 4.4 W/kg). Microwaves alone had no effect on transformation. Plating efficiency and cell survival were not affected by TPA or microwave treatments.     

Cell-cycle-dependent radiation-induced oncogenic transformation of C3H 10T1/2 cells.

C3H 10T1/2 cells were synchronized by a modified mitotic shake-off procedure. X irradiation of cells at various intervals after mitotic harvest indicated a single narrow window (about 2 h) of sensitivity to the induction of oncogenic transformation. It is not possible to delineate precisely the time in the cycle at which this sensitivity is expressed. The most likely candidate is G2 phase, though we cannot eliminate the possibility that the sensitive period begins in late S phase. In the same synchronized cells, cell lethality showed the conventional pattern, i.e., sensitivity in mitosis and resistance in late S and in G1 phase.     

Radiosensitivity parameters for neoplastic transformations in C3H10T1/2 cells

We have evaluated radiosensitivity parameters for cellular transformation from published experimental data on neoplastic transformations induced in C3H10T1/2 cells by BEVALAC ions. The measured RBE values are well reproduced by a track theory calculation using sets of m-target parameters with either m = 2 or m = 3, suggesting a quadratic or cubic extrapolation to low doses of gamma rays. Using track theory one is thus able to predict transformation frequencies in those cells after an arbitrary radiation field, under known or assumed conditions of exposure, in a manner shown earlier for cellular survival. Extension of these calculations to interpret cancer incidence in vivo is also discussed.     

Neutron-energy-dependent oncogenic transformation of C3H 10T1/2 mouse cells

The relative biological effectiveness (RBE) of a range of neutron energies relative to 250-kVp X rays has been determined for oncogenic transformation and cell survival in the mouse C3H 10T 1/2 cell line. Monoenergetic neutrons at 0.23, 0.35, 0.45, 0.70, 0.96, 1.96, 5.90, and 13.7 MeV were generated at the Radiological Research Accelerator Facility of the Radiological Research Laboratories, Columbia University, and were used to irradiate asynchronous cells at low absorbed doses from 0.05 to 1.47 Gy. X irradiations covered the range 0.5 to 8 Gy. Over the more than 2-year period of this study, the 31 experiments provided comprehensive information, indicating minimal variability in control material, assuring the validity of comparisons over time. For both survival and transformation, a curvilinear dose response for X rays was contrasted with linear or nearly linear dose responses for the various neutron energies. RBE increased as dose decreased for both end points. Maximal RBE values for transformation ranged from 13 for cells exposed to 5.9-MeV neutrons to 35 for 0.35-MeV neutrons. This study clearly shows that over the range of neutron energies typically seen by nuclear power plant workers and individuals exposed to the atomic bombs in Japan, a wide range of RBE values needs to be considered when evaluating the neutron component of the effective dose. These results are in concordance with the recent proposals in ICRU 40 both to change upward and to vary the quality factor for neutron irradiations.     

p21ras and protein kinase C function in distinct and interdependent signaling pathways in C3H 10T1/2 fibroblasts.

Both p21ras and protein kinase C (PKC) are believed to function downstream of plasma membrane-associated tyrosine kinases in cellular signal transduction pathways. However, it has remained controversial whether they function in the same pathway and, if so, what their relative position and functional relationship in such a pathway are. We investigated the possibilities that p21ras and PKC function either upstream or downstream of each other in a common linear pathway or that they function independently in colinear signal pathways. Either decreased expression of endogenous normal ras in fibroblasts transfected with an inducible antisense ras construct or overexpression of a mutant ras gene reduced the capacity of the phorbol ester tetradecanoyl phorbol acetate to trigger expression of the tetradecanoyl phorbol acetate-responsive and ras-dependent reporter gene osteopontin (OPN). PKC depletion decreased basal OPN mRNA levels, and the overexpression of ras restored OPN expression to the level of non-PKC-depleted cells. We propose a model in which ras and PKC function in distinct and interdependent signaling pathways.     

Absence of a dose-fractionation effect on neoplastic transformation induced by fission-spectrum neutrons in C3H 10T1/2 cells

We have investigated the effect of fission-spectrum neutron dose fractionation on neoplastic transformation of exponentially growing C3H 10T1/2 cells. Total doses of 10.8, 27, 54, and 108 cGy were given in single doses or in five equal fractions delivered at 24-h intervals in the biological channel of the RSV-TAPIRO reactor at CRE-Casaccia. Both cell inactivation and neoplastic transformation were more effectively induced by fission neutrons than by 250-kVp X rays. No significant effect on cell survival or neoplastic transformation was observed with split doses compared to single doses of fission-spectrum neutrons. Neutron RBE values relative to X rays determined from data for survival and neoplastic transformation were comparable.     

Ribozyme-mediated perlecan knockdown impairs chondrogenic differentiation of C3H10T1/2 fibroblasts

Perlecan (Pln) is an abundant heparan sulfate (HS) proteoglycan in the pericellular matrix of developing cartilage, and its absence dramatically disrupts endochondral bone formation. This study examined two previously unexamined aspects of the function of Pln in mesenchymal chondrogenesis in vitro. Using the well-established high-density micromass model of chondrogenic differentiation, we first examined the requirement for endogenous Pln synthesis and secretion through the use of Pln-targeted ribozymes in murine C3H10T1/2 embryonic fibroblasts. Second, we examined the ability of the unique N-terminal, HS-bearing Pln domain I (PlnDI) to synergize with exogenous bone morphogenetic protein-2 (BMP-2) to support later stage chondrogenic maturation of cellular condensations. The results provide clear evidence that the function of Pln in late stage chondrogenesis requires Pln biosynthesis and secretion, because 60%-70% reductions in Pln greatly diminish chondrogenic marker expression in micromass culture. Additionally, these data support the idea that while early chondrocyte differentiation can be supported by exogenous HS-decorated PlnDI, efficient late stage PlnDI-supported chondrogenesis requires both BMP-2 and Pln biosynthesis.     

Molecular phenotyping of non-transformed and chemically transformed C3H10T1/2 mouse fibroblasts

A systematic molecular phenotyping approach based on two-dimensional gel electrophoresis is being applied in an attempt to identify protein changes associated with malignant transformation. Using the C3H10T1/2 mouse cell line, two-dimensional polypeptide maps of the non-transformed cell line, several chemically transformed lines and a tumour cell line were compared. Although there is a large degree of similarity between the protein profiles of all cell lines, clear differences are evident. Initial results are consistent with the view that many of the protein changes are incidental to malignant transformation. Changes induced by 3-methylcholanthrene are retained after transplantation of the cells into nude mice.     

Modification of transformation of C3H/10T1/2 cells by a differentiation-inducing substance

Transformation of C3H/10T1/2 cells was induced by 3-methylcholanthrene. Treatment with hexamethylene bisacetamide (HMBA), a differentiation inducing and poly (ADP-ribose)-synthesis modifying substance, influences expression of multilayered foci in a treatment schedule-dependent manner. Inhibition of transformation occurred only if HMBA was present after the genotoxic damage. After HMBA treatment most of transformed cells showed an end-differentiation like form.     

In vitro transformation by bromodeoxyuridine and X irradiation in C3H 10T1/2 cells

The effect of 10(-5) M bromodeoxyuridine (BrdUrd) substitution in C3H 10T1/2 cells was evaluated. Cellular toxicity increased rapidly for BrdUrd exposure times that were longer than the population doubling time. Radiosensitization by BrdUrd exposure was almost complete after one cell doubling time and was characterized by a decrease in D0 and the survival curve shoulder. Exposure to BrdUrd for one cell doubling time produced only very low transformation levels, but for prolonged BrdUrd exposure times, the transformation frequency per viable cell increased significantly. BrdUrd incorporation also enhanced radiation induction of transformation above the transformation levels resulting from the independent action of X rays or BrdUrd treatment. These results show that BrdUrd is a transforming agent in C3H 10T1/2 cells and thus may be a carcinogen and that BrdUrd can enhance radiation-induced transformation.     

Morphological transformation of C3H/10T1/2 cells subcultured at low cell densities

Morphological transformation in $\text{C3H/10T}\text{1}\text{2}$ cells treated with varied concentrations of benzo (α) pyrene (BP) was measured following subculture at low cell densities. Subconfluent cultures exposed to BP were allowed to grow to confluence, trypsinized, and reseeded at cell densities ranging from 5 to 2,300 surviving cells/cm². These secondary cultures were incubated for 8 to 9 weeks, stained, and examined for evidence of morphological transformation. BP-treated cells reseeded in virtual isolation in microwells (approx. 5 surviving cells/cm²) transformed at frequencies up to 14.5%. At these low initial cell densities, transformation frequency did not demonstrate a significant dependence on BP concentration. However, BP-treated cells reseeded at higher densities (11 to 2,300 surviving cells/cm²) showed both density-dependent transformation frequencies and BP-concentration dependence of transformation. As reported previously (Haber et al., Cancer Res. 37 1644, 1977), the subculturing of treated cells did not affect the BP-concentration dependence of focus formation in the $\text{C3H/10T}\text{1}\text{2}$ transformation assay. Cell density-dependent suppression of morphological transformation has now been observed over a wide range of BP concentration. We suggest that this phenomenon is associated with colony interactions and consider various possible mechanisms of BP involvement.     

DNA binding of aflatoxin B1 by co-oxygenation in mouse embryo fibroblasts C3H/10T1/2

The mechanism of activation of aflatoxin B1 to ultimate metabolites capable of DNA binding was investigated in mouse embryo fibroblasts C3H/10T1/2. The contribution of co-oxygenation reactions which are coupled to arachidonic acid metabolism was assessed by the use of inhibitors of prostaglandin endoperoxide synthetase and lipoxygenase. Indomethacin and 5,8,11,14-icosatetraynoic acid inhibited AFB1-binding to maximally 60%. The antioxidant glutathione was also inhibitory while CuZn superoxide dismutase had no effect or slightly stimulated binding at high concentrations. These results indicate that co-oxygenation plays a major role in AFB1-metabolism in 10T1/2 cells. The observation that the phospholipase A2 inhibitor p-bromophenacylbromide diminished AFB1-DNA binding supports the notion that AFB1, because it is membrane-active, may enhance its own co-oxidative metabolism by stimulating the arachidonic acid cascade.     

Lactate-mediated changes in growth morphology and transformation frequency of irradiated C3H 10T1/2 cells

Treatment of mammalian cells with lactate or inhibitors of glycolysis alters their radiation response, particularly in the low dose region of the dose response curve. The occurrence of both high lactate levels and high glycolytic metabolism in tumours is well known and therefore the effect of lactate on a cell line sensitive to radiation induced transformation was examined using a single exposure to Cobalt 60 gamma rays as the carcinogen challenge. The results indicate that cells treated with 5mM lactate before irradiation exhibit changes in morphology and growth rate and that the transformation frequency is increased by three to ten fold following 24 hours lactate treatment just prior to irradiation. Examination of radiation survival curves showed a positive correlation between transformation frequency and size of the shoulder, but increasing transformation frequency was associated with a decrease in Do. A mechanism involving altered Redox potential in lactate treated cells is suggested. The results are discussed in terms of their possible significance for radiotherapy.     

Morphological transformation and chromosome damage by amsacrine in C3H/10T1/2 clone 8 cells

Morphological transformation, cell survival, chromosomal aberrations and micronuclei were measured in C3H/101/2CL8 cells after 24 h exposure to amsacrine. A weak but dose-related increase in the percentage of dishes containing transformed foci occurred. As previously reported for alkylating agents, this effect was increased by treating 5 days instead of 1 day after plating. There was no evidence for gene mutation at the Na/K ATPase locus, although amsacrine induced micronuclei in a large percentage of cells and chromosomal aberrations, including interchange events and double minute chromosomes, in dividing cells. In would appear that transformation and chromosomal events may be related in amsacrine-treated C3H/10T1/2CL8 cells. The results strongly suggest that amsacrine has carcinogenic potential, possibly related to its chromosome-breaking properties.     

Retinyl acetate inhibits platelet-derived growth factor-induced Ca2+ signals in C3H 10T1/2 fibroblasts

Mitogenic stimulation of density-arrested C3H 10T1/2 mouse fibroblasts by serum or purified platelet-derived growth factor (PDGF) was potently inhibited by retinyl acetate (RAc; IC50 = 0.1 microgram/ml, 0.3 x 10(-6) M) when administered during the first 2 hours of mitogen exposure. This inhibitory effect of RAc coincided with a period early in the cell growth-division cycle when density-arrested C3H 10T1/2 cells stimulated by PDGF were found to require physiological levels of extracellular Ca2+ for the transition from G0 to G1 of the cell cycle. To determine if the inhibitory effect of RAc was mediated through alterations in the Ca2+ signaling pathway induced by mitogens, we examined Fura-2-loaded fibroblasts for changes in the Ca2+ response elicited by PDGF. Addition of PDGF (5 ng/ml) induced a transient increase in the [Ca2+]i that was not significantly effected by the extracellular Ca2+ concentration. Treatment of cells with RAc caused a concentration- and time-dependent inhibition of this PDGF-stimulated Ca2+ flux (IC50 = 0.45 microgram/ml or 1.5 x 10(-6) M; t1/2 = 15 min), whereas release of intracellularly stored Ca2+ by thrombin was unaffected by RAc (1.2 micrograms/ml, 4 x 10(-6) M). Treatment with RAc did not significantly affect PDGF binding to cell surface receptors or the generation of inositol phosphates. These results suggest that the mechanism by which RAc inhibits PDGF- or serum-induced mitogenesis is through modulation of the Ca2+ signal stimulated by PDGF, and thereby depriving the cell of a rise in intracellular Ca2+ necessary for progression through the cell cycle.